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The ETV6-NTRK3 gene fusion encodes a chimeric protein tyrosine kinase that transforms NIH3T3 cells

Oncogene volume 19, pages 906–915 (2000)Cite this article

Abstract

The congenital fibrosarcoma t(12;15)(p13;q25) rearrangement splices the ETV6 (TEL) gene on chromosome 12p13 in frame with the NTRK3 (TRKC) neurotrophin-3 receptor gene on chromosome 15q25. Resultant ETV6-NTRK3 fusion transcripts encode the helix–loop–helix (HLH) dimerization domain of ETV6 fused to the protein tyrosine kinase (PTK) domain of NTRK3. We show here that ETV6-NTRK3 homodimerizes and is capable of forming heterodimers with wild-type ETV6. Moreover, ETV6-NTRK3 has PTK activity and is autophosphorylated on tyrosine residues. To determine if the fusion protein has transforming activity, NIH3T3 cells were infected with recombinant retroviral vectors carrying the full-length ETV6-NTRK3 cDNA. These cells exhibited a transformed phenotype, grew macroscopic colonies in soft agar, and formed tumors in severe combined immunodeficient (SCID) mice. We hypothesize that chimeric proteins mediate transformation by dysregulating NTRK3 signal transduction pathways via ligand-independent dimerization and PTK activation. To test this hypothesis, we expressed a series of ETV6-NTRK3 mutants in NIH3T3 cells and assessed their transformation activities. Deletion of the ETV6 HLH domain abolished dimer formation with either ETV6 or ETV6-NTRK3, and cells expressing this mutant protein were morphologically non-transformed and failed to grow in soft agar. An ATP-binding mutant failed to autophosphorylate and completely lacked transformation activity. Mutants of the three NTRK3 PTK activation-loop tyrosines had variable PTK activity but had limited to absent transformation activity. Of a series of signaling molecules well known to bind to wild-type NTRK3, only phospholipase-Cγ (PLCγ) associated with ETV6-NTRK3. However, a PTK active mutant unable to bind PLCγ did not show defects in transformation activity. Our studies confirm that ETV6-NTRK3 is a transforming protein that requires both an intact dimerization domain and a functional PTK domain for transformation activity.

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Acknowledgements

The authors would like to thank P Marynen for providing the ETV6 cDNA and α-ETV6:HLH antibody, D Kaplan for helpful discussions and for providing NIH3T3 cells expressing NTRK3, B Nelkin for providing the NTRK3 cDNA and NTRK3-overexpressing medullary thyroid carcinoma cells, and C Denny for providing EWS-FLI1 constructs for transformation of NIH3T3 cells. We also thank Jerian Lim for expert technical work. This work was supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada (with funds from the Terry Fox Run) to Dr P Sorensen, and from the Medical Research Council of Canada and the National Cancer Institute of Canada to Dr R Kay.

The updated full-length ETV6-NTRK3 cDNA sequence is deposited in Genbank under accession number AF041811.

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  1. Department of Pathology, 4480 Oak St., British Columbia's Children's Hospital, Vancouver, V6H 3V4, British Columbia, Canada

    Daniel H Wai, Stevan R Knezevich & Poul HB Sorensen

  2. Department of Clinical Pharmacology, Section of Experimental Oncology/Molecular Pharmacology, University of Vienna, Waehringer Guertel 18-20, Vienna, A-1090, Austria

    Trevor Lucas & Burkhard Jansen

  3. Department of Dermatology, Division of General Dermatology, University of Vienna, Waehringer Guertel 18-20, Vienna, A-1090, Austria

    Trevor Lucas & Burkhard Jansen

  4. Department of Medical Genetics, Terry Fox Laboratory, British Columbia Cancer Agency, 600 W. 10th Avenue, Vancouver, V5Z 4E6, British Columbia, Canada

    Robert J Kay

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Wai, D., Knezevich, S., Lucas, T. et al. The ETV6-NTRK3 gene fusion encodes a chimeric protein tyrosine kinase that transforms NIH3T3 cells. Oncogene 19, 906–915 (2000). https://doi.org/10.1038/sj.onc.1203396

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